Project Summary/Abstract KS is considered an AIDS-defining malignancy; however, there is still debate about whether KS is a reactive process or a true clonal cancer. Several features of KS suggest that it may not result from a transformation event that results in autonomously growing tumor cells, but represents a reactive process due to the combined effects of a virus with angiogenic properties and local or systemic inflammation. However, KS is very heterogeneous in terms of its histological, epidemiological and clinical features, and mounting evidence suggests that there may be different subtypes. KS is consistently associated with Kaposi sarcoma-associated herpesvirus (KSHV) infection, and this virus is now well documented to be the etiologic agent of KS. Therefore, a large number of studies have focused on understanding KSHV and the viral mechanisms of pathogenesis. Yet, there is a dearth of information about any cellular genomic alterations occurring in KS. These cellular genomic alterations characterize most other cancers, including those associated with infection by other human oncogenic viruses. Our preliminary sequencing studies on KS tumors have identified recurrent genomic losses of a tumor suppressor gene, WT1 as well as mutations in two proteins of the anaphase promoting complex (APC/C), involved in important cellular functions and cancer development. Moreover, our data suggest that some of these genetic alterations are clonal in some KS tumors. Building on these observations, we now propose to perform the first comprehensive analysis of cellular genetic alterations in KS on a large sample of clinically-annotated tumors. We will use state-of-the-art deep sequencing approaches to determine whether cellular genetic alterations exist in KS tumors, which genes are most commonly affected, and whether these mutations are monoclonal in some or all cases. We will also explore the relationship of mutational burden, specific gene mutations and clonality with KS clinical presentation and response to therapy. Through this approach, we will test the hypothesis that some KS lesions are polyclonal reactive processes largely driven by KSHV, and others full-blown malignancies with clonal genetic alterations in biologically relevant genes. We will test this hypothesis through two specific aims: 1) To determine whether all or a subset of KS tumors have recurrent genetic alterations in known cancer-related genes or other regions of the coding cellular genome; and 2) To characterize the relationship between tumor genomic alterations and KS clinical presentation and outcomes. By determining whether KS tumors possess genetic mutations within clonal cell populations, this study will contribute significantly to our basic understanding of disease pathogenesis, and could ultimately have important implications for disease staging and treatment.